NEW HIGHLY EFFECTIVE
DRY POWDER TOBRAMYCIN FORMULATIONS FOR INHALATION IN THE TREATMENT
OF CYSTIC FIBROSIS
GABRIELLE PILCER
Pharmacien
Thèse présentée en vue de l’obtention du grade de Docteur en Sciences Biomédicales et Pharmaceutiques
INSTITUT DE PHARMACIE
Laboratoire de Pharmacie Galénique et de Biopharmacie Professeur K. AMIGHI
Monsieur le Professeur Karim Amighi pour m’avoir accueillie au sein de son Laboratoire de Pharmacie Galénique et de Biopharmacie. Les moyens qu’il a mis en œuvre pour que ce travail se déroule dans les meilleures conditions, son expérience, sa compétence, sa rigueur scientifique, ses nombreux conseils, sa constante disponibilité et son ouverture d’esprit ont rendu ces 4 années de doctorat, passées à ses côtés, particulièrement enrichissantes et agréables. Je le remercie également pour la confiance et la liberté qu’il m’a accordées ainsi que pour la qualité de la formation qu’il m’a offerte. Qu’il me soit permis ici de lui exprimer toute ma reconnaissance et mon profond respect.
Je tiens également à exprimer mes très sincères remerciements à SMB Galéphar pour le support financier et logistique octroyé.
Je voudrais tout particulièrement remercier le Docteur Francis Vanderbist, Directeur du Département Recherche et Développement, pour sa disponibilité, son chaleureux soutien ainsi que pour les nombreux échanges scientifiques que nous avons eus lors de nos réunions.
Je tiens aussi à remercier, pour leur accueil amical, tout le personnel de ce département avec une attention spéciale pour Fabrice Van Kerkhoven et Renilde Sibenaler. La patience et les compétences, dont ils ont fait preuve pour la mise au point et la validation de la méthode LC/MS-MS de dosage de la tobramycine ainsi que pour les analyses pharmacocinétiques lors de l’étude clinique, m’ont été particulièrement utiles.
Je tiens également à exprimer ma gratitude aux Docteurs Serge Goldman et Didier Blocklet du Service des Radio-Isotopes – Médecine Nucléaire de l’Hôpital Erasme pour leur participation à l’étude clinique.
Un tout grand merci, aussi, au Docteur Christiane Knoop et au kinésithérapeute Christian Opdekamp, du Service de Pneumologie, pour leur aide. Leur dévouement et leur implication, vis-à-vis des patients atteints de mucoviscidose, a été une source de motivation supplémentaire tout au long de ce travail.
Plus particulièrement, j’adresse mes remerciements les plus chaleureux au Pharmacien Bernard Van Gansbecke (Service des Radio-Isotopes et de Pharmacie Hospitalière) pour son aide et soutien inestimables durant l’étude clinique. Son professionnalisme, sa disponibilité et son savoir transmis avec enthousiasme ont rendu le travail de marquage radioactif des formulations de tobramycine passionnant.
Industrielle de l’Université Libre de Bruxelles, pour leur collaboration aux analyses de diffraction aux rayons X et de microscopie électronique à balayage ainsi que pour leur entière disponibilité.
Merci à Nancy Van Aelst et Philippe Deleuze, les deux techniciens du service de Pharmacie Galénique et de Biopharmacie, sans lesquels la vie au laboratoire ne serait pas la même.
De même, j’en profite également pour adresser mes remerciements aux anciens doctorants du service pour leur chaleureux accueil à mes débuts: Jamila Hamdani, Jérôme Hecq et Thami Sebti.
J’aimerais aussi exprimer le réel plaisir que je ressens à côtoyer quotidiennement les doctorantes Flore Depreter, Laurence Durand et Nathalie Wauthoz. La bonne entente qui règne entre nous et les liens d’amitiés qui se sont crées ont certainement contribué à rendre l’ambiance propice à la bonne réalisation de ce travail.
Et enfin, je tiens à remercier celui avec qui j’ai débuté et terminé cette aventure, Jonathan Goole, dont la gentillesse et la serviabilité m’ont constamment accompagnée et m’ont permis d’apprécier la force de son amitié. De partager nos avancées et nos difficultés respectives et de suivre de manière continue la progression de nos travaux a permis de tisser entre nous une grande complicité.
Un tout grand merci pour tous les membres de l’Institut de Pharmacie que j’ai eu le plaisir de croiser ces dernières années. Et plus particulièrement, merci à Pierre Van Antwerpen et Marie Faes pour leurs précieux conseils et leur inébranlable bonne humeur.
De manière plus personnelle, mes pensées se tournent vers l’ensemble de ma famille et de mes amis pour leur écoute attentive, leur soutien sans faille et tous les bons moments passés ensemble. Plus particulièrement, je remercie mes parents pour l’affectueuse attention dont ils m’entourent et pour m’avoir transmis le plaisir d’apprendre.
TABLE OF CONTENTS
ABBREVIATIONS 8
I. SUMMARY 9
II. INTRODUCTION 14
II.1. DRUG DELIVERY ROUTES TO THE LUNGS 15
II.1.1. Oral drug delivery 15
II.1.2. Parenteral drug delivery 16
II.1.3. Pulmonary drug delivery 16
II.2. CHARACTERISTICS OF THE LUNG 18
II.2.1. Anatomy of the airways 18
II.2.2. Physiology of the airways 22
II.2.3. Blood supply 24
II.2.4. Lung volumes 25
II.3. PULMONARY DEPOSITION 26
II.3.1. Deposition mechanisms of inhaled particles 26
II.3.2. Influence of ventilatory parameters 27
II.3.3. Influence of respiratory tract morphology 28 II.3.4. Influence of aerosol characteristics: diameter 29
II.4. DELIVERY DEVICES 30
II.4.1. Nebulizers 30
II.4.2. Pressurized Metered-Dose Inhalers 33
II.4.3. Dry Powder Inhalers 36
II.4.3.1. Unit-dose devices 40
II.4.3.2. Multi-dose devices 42
II.4.3.3. Deaggregation and deagglomeration 44 II.4.3.4. Importance of the inspiratory airflow 45
II.4.3.5. Compliance of patients 47
II.4.3.6. Future research and new developments 48
II.5. FORMULATION OF DRY POWDER INHALERS 49
II.5.1. Types of formulation 49
II.5.1.1. Excipients 49
II.5.1.1.1. Nature of excipients 49
II.5.1.1.2. Blending 52
II.5.1.2. Liposomes 53
II.5.1.3. Cyclodextrins 54
II.5.1.4. Biodegradable microspheres 54
II.5.1.5. Large porous particles 56
II.5.1.6. Carrier-free 56
II.5.2. Forces of interaction 57
II.5.2.1. Surface morphology 58
II.5.2.2. Moisture content and hygroscopicity 59
II.6. MICRONIZATION 61
II.6.1. Milling 61
II.6.2. Spray drying 63
II.6.3. Supercritical fluids 66
II.7. CYSTIC FIBROSIS 68
II.7.1. Disease 68
II.7.2. Treatments 70
II.7.2.1. Clearance of airway secretions 71
II.7.2.1.1. Chest physiotherapy 71
II.7.2.1.2. Mucolytics 71
II.7.2.2. Treatment of pulmonary infections: Antibacterial therapy 73
II.7.2.3. Lung transplantation 75
II.7.2.4. Pancreatic enzyme replacements 75
II.7.2.5. Future therapies 75
II.7.2.5.1. Gene therapy 75
II.7.2.5.2. Pharmacological modulation of ion transport 76
III. AIM OF THE WORK 77
IV. MATERIALS AND METHODS 80
IV.1. MATERIALS 81
IV.2. METHODS 81
IV.2.1. High speed homogenization 81
IV.2.2. High pressure homogenization 82
IV.2.3. Spray drying 83
IV.2.3.1. Preparation of formulations containing nanoparticles 84 IV.2.3.2. Preparation of carrier-free formulations 84 IV.2.3.3. Preparation of lipid-coated particles 85
IV.2.4. Scanning electron microscopy 86
IV.2.5. X-ray powder diffraction 87
IV.2.6. Differential scanning calorimetry 87
IV.2.7. Bulk density 88
IV.2.8. Determination of water content 89
IV.2.9. Laser diffraction 90
IV.2.9.1. Mastersizer 2000® 91
IV.2.9.2. Spraytec® 92
IV.2.10. Unifomity of delivered dose 93
IV.2.11. Uniformity of drug content 94
IV.2.12. Assessment of lung deposition 94
IV.2.12.1 In vitro assessment of lung deposition 94
IV.2.12.1.1. Impactors and impingers 94
IV.2.12.1.1.1. Glass impinger 96
IV.2.12.1.1.2. Andersen cascade impactor 96
IV.2.12.1.1.3. Multi-stage liquid impinger 96
IV.2.12.1.1.4. New generation impactor 97
IV.2.12.1.2. Interpretation of results 99
IV.2.12.1.3. Aerodynamic particle size analysis 100
IV.2.12.1.3.1. Derivatization procedure 101
IV.2.12.1.3.2. HPLC system 102
IV.2.12.2. In vivo assessment 103
IV.2.12.2.1. Pharmacokinetic analysis 103
IV.2.12.2.2. Gamma scintigraphy analysis 104
IV.2.12.2.3. Radiolabelling method 106
IV.2.12.2.4. Validation of radiolabelled powder 106
IV.2.13. Statistical analysis 107
V. EXPERIMENTAL PART 109
PART V.1: CORRELATIONS BETWEEN CASCADE IMPACTOR ANALYSIS AND LASER DIFFRACTION TECHNIQUES FOR THE DETERMINATION
OF THE PARTICLE SIZE OF AEROSOLIZED POWDER FORMULATIONS 110
V.1.1. Introduction 111
V.1.2. Results 112
V.1.2.1. Particle size distribution by laser diffraction 113 V.1.2.2. Particle size distribution by inertial impaction 116 V.1.2.3. Correlation between inertial impaction and Spraytec® laser diffraction 117
V.1.2.5. Evaluation of influence of the flow rate 121 V.1.2.6. Evaluation of maximal capacity of the capsule 123
V.1.3. Discussion 124
V.1.4. Conclusion 126
PART V.2: FORMULATION AND CHARACTERIZATION OF LIPID-COATED
TOBRAMYCIN PARTICLES FOR DRY POWDER INHALATION 128
V.2.1. Introduction 129
V.2.2. Results and discussion 130
V.2.2.1. Physicochemical characteristics 130
V.2.2.2. Aerodynamic behaviour 136
V.2.3. Conclusion 140
PART V.3: PREPARATION AND CHARACTERIZATION OF SPRAY-DRIED TOBRAMYCIN POWDERS CONTAINING NANOPARTICLES
FOR PULMONARY DELIVERY 141
V.3.1. Introduction 142
V.3.2. Results and discussion 144
V.3.2.1. Preparation of nanosuspensions 144
V.3.2.1.1. Formulation composition 144
V.3.2.1.2. Influence of pre-homogenization and HPH operations 146
V.3.2.2. Evaluation of dry powder formulations 148
V.3.2.2.1. Physicochemical characteristics 149
V.3.2.2.2. Aerodynamic behaviour 154
V.3.3. Conclusion 157
PART V.4: SPRAY-DRIED CARRIER-FREE DRY POWDER TOBRAMYCIN
FORMULATIONS WITH IMPROVED DISPERSION PROPERTIES 159
V.4.1. Introduction 160
V.4.2. Results and discussion 161
V.4.2.1 Influence of spray-drying temperature 161
V.4.2.2. Influence of amount of water in the suspensions 166
V.4.3. Conclusion 173
PART V.5: PHARMACOSCINTIGRAPHIC AND PHARMACOKINETIC EVALUATION OF TOBRAMYCIN DPI FORMULATIONS IN CYSTIC FIBROSIS PATIENTS 175
V.5.1. Introduction 176
V.5.2. Study protocol 176
V.5.2.1. Dry Powder Formulations 176
V.5.2.2. Study design 177
V.5.2.3. Subjects 179
V.5.2.4. Safety assessment 180
V.5.3. Results 181
V.5.3.2. Lung function tests 182
V.5.3.3. Scintigraphic results 184
V.5.3.4. Pharmacokinetic data 190
V.5.4. Discussion 192
V.5.5. Conclusion 194
PART V.6: STABILITY STUDIES 195
V.6.1. Introduction 196
V.6.2. Results 199
V.6.2.1. XRPD analyses 199
V.6.2.2. Uniformity of delivered dose 202
V.6.2.3. Particle size distribution and in vitro deposition 203
V.6.3. Conclusion 211
VI. GENERAL CONCLUSION 212
VII. REFERENCES 217
VIII. ANNEXES 247
VIII.1. Validation criteria of the LC/M-MS method 248
VIII.2. Ethics committee approval 249
VIII.3. Directorate-General for Medicinal Products Approval 251
VIII.4. Declaration of Helsinki 252
VIII.5. Patient information 257
VIII.6. Patient declaration 261
ABBREVIATIONS
99mTc Sodium Pertechnetate KF Karl Fisher titration method ACI Andersen Cascade Impactor HPH High Pressure Homogenization AUC Area Under the Curve HPLC High Pressure Liquid Chromatography Cmax Maximal plasma concentration HPMC Hydroxypropyl Methyl Cellulose CF Cystic Fibrosis HSH High Speed Homogenization CFC Chlorofluorocarbon ICH International Conference on
Harmonisation CFTR Cystic Fibrosis Transmembrane MDI Metered Dose Inhaler
Conductance Regulator
COPD Chronic Obstructive Pulmonary Disease MMAD Mass Median Aerodynamic Diameter CV Coefficient of Variation MOC Micro-Orifice Collector
Daer Aerodynamic Diameter MsLI Multi-stage Liquid Impinger Dgeo Geometric Diameter NAL Nacystelyn
DPI Dry Powder Inhaler NGI New Generation Impactor
DPPC Dipalmitoylphosphatidylcholine PLGA Poly(lactic-co-glycolic) acid polymer DSC Differential Scanning Calorimetry PSD Particle Size Distribution
EDTA Ethylene Diamine Tetraacetic Acid RH Relative Humidity FEV1 Forced Expiratory Volume in one second SCF Supercritical Fluid FDA Food and Drug Administration SD Standard Deviation
FPD Fine Particle Dose SEM Scanning Electron Microscopy FPF Fine Particle Fraction S.E.M. Standard Error Mean
FVC Forced Vital Capacity Tmax Time to reach maximum plasma concentration
GSD Geometric Standard Deviation TGA Thermogravimetric Analysis HFA Hydrofluoroalkanes XRPD X-Ray Powder Diffraction
I. SUMMARY
I. SUMMARY
Local delivery of medication to the lung is highly desirable as the principal advantages include reduced systemic side effects and higher dose levels of the applicable medication at the site of drug action. This administration could be particularly useful for patients with specifically chronic pulmonary infections or pulmonary diseases, such as cystic fibrosis, asthma or lung cancer.
In order to deliver a high dose range of medication for highly-dosed drugs such as antibiotics, “carrier-free” DPI formulations of tobramycin were developed with the aim of minimizing the use of excipients. Briefly, dry powders were prepared by spray drying various suspensions of tobramycin in isopropanol.
First, as particle size is a key parameter in defining drug deposition in the lungs, the new Spraytec® laser diffraction method specifically modified for measuring the PSD of aerosolized drug was evaluated. The dispersion properties of various dry powder formulations were investigated using different laser diffraction and impaction apparatuses at different flow rates and using different inhalator devices. Different correlations between geometric and aerodynamic size data were demonstrated in this study. As a potential application, for the flow rate, the different inhalation devices and the drug formulations examined, the tobramycin fine particle fraction could be predicted from measurements obtained from the Spraytec® using linear relationships. Correlations (R² > 0.9) between the MMAD and the percentage of particles with a diameter below 5 µm could be demonstrated between the results obtained from the laser diffraction technique and the impaction method.
Consequently, the Spraytec® laser diffraction technique was proved to be an important tool for initial formulation and process screening during formulation development of DPIs.
In order to modify the surface properties of the raw tobramycin powder, different powder compositions were formulated with the aim of studying the influence of the concentration of tobramycin in drug suspensions used for spray-drying, the lipid film composition (cholesterol:Phospholipon ratio) and the coating level (in percentage) on the
The results indicated that the application of a lipid coating around the active particles allowed an improvement in particle dispersion from the inhalator, decreasing raw powder agglomeration and thus enhancing drug deposition deep in the lungs. Moreover, these results seemed to be influenced by the amount and composition of the lipids in the formulations. The evaluation of the influence of the coating level showed that the deposition of only 5% w/w lipids (on a dry basis) was sufficient to improve particle dispersion properties during inhalation. The FPF, which is around 36% for the uncoated micronized tobramycin, was increased to up to about 68% for the most effective lipid-coated formulation. Of particular importance, these results revealed the need to add sufficient amounts of covering material in order to significantly modify the particle surface properties and reduce their tendency to agglomeration, while limiting the lipid level in the formulations in order to avoid any undesirable sticking and to allow the delivery of more of the active drug to the deep lung.
Another approach used to modify the surface properties of raw tobramycin was to coat the micronized particles with nanoparticles of the drug, produced by high pressure homogenization. The evaluation of the influence of the level of nanoparticle coating of the micronized particles showed that the presence of nanoparticles in the formulations improved the particle dispersion properties during inhalation. One microparticle was completely covered with a single layer or several layers of nanoparticles, in function of the percentage of nanoparticles in the mixture. Coating the fine drug particles with particles in the nanometer range was believed to reduce Van Der Waals forces and powder agglomeration. These various layers of nanoparticles also allowed a decrease in the cohesion of the powder by improving the slip between the particles.
On the other hand, suspensions containing solely nanoparticles were spray dried with various concentrations of surfactant in order to produce easily dispersible and reproducible micron-size agglomerates of nanoparticles during inhalation. The evaluation of the influence of the concentration of surfactant showed that deposition of only 2% w/w (on a dry basis) of Na glycocholate is sufficient to improve particle dispersion properties during inhalation.
Consequently, the use of nanoparticles in dry powder formulations increased the FPF from 36% for the uncoated micronized tobramycin to about 61% for this latter formulation.
To modify the balance between the different forces of interactions without the need for any excipient, the influence of formulation components on the aerosolization characteristics of spray-dried tobramycin through the use of various proportions of water in the solvent used to prepare initial suspensions was investigated. These results showed that it is possible to modify the surface properties of the particles by coating the particles of drug with a homogeneously distributed film of the active compound dissolved in a solvent system containing a mixture of different solvents such as isopropanol and water. During nebulization of the suspension, droplets are composed of one or more particles in solid state surrounded with solvent containing the dissolved drug. It is hypothesized that during the drying step, dissolved tobramycin forms a coating of the amorphous drug around particles in suspension. The coating of drug particles can thus be used as an alternative approach that permits the modification of the surface properties of the particles, increasing the flowability, the desagglomeration tendency and the fine particle fraction deposited in the deep lung. So, the evaluation of the influence of the water content of the suspensions and the effect of the inlet temperature during spray-drying showed that the addition of 2% water v/v is sufficient to improve particle dispersion during inhalation. Of particular interest, as tobramycin is a very hygroscopic drug, the addition of water turned out to be a critical step. It was thus important to add a small amount of water to the solvent system and to process the drying step at a high temperature to produce formulations containing solely the active drug and showing a FPF of up to 50%.
Moreover, stability studies demonstrated that these optimized formulations (lipid- coated formulation, nanoparticle formulation and amorphous drug-coated formulation) were stable over a long time period at various ICH temperature and relative humidity storage conditions (25°C/60% RH, 30°C/65% RH and 40°C/75% RH). The formulations were shown to keep their crystalline state, initial PSD, redispersion characteristics and deposition results for more than twelve months.
In order to confirm these encouraging results, two optimized formulations (one with a lipid coating and another with amorphous drug coating) were selected and compared to
solution), by performing a combined in vivo scintigraphic and pharmacokinetic evaluation of tobramycin DPIs in nine CF patients.
In comparison with Tobi®, it was estimated that lung deposition, expressed as a percentage of the nominal dose, was 7.0 and 4.5 times higher for the lipid-coated and amorphous tobramycin-coated formulations, respectively. Moreover, the pharmacokinetic data, adjusted to the same drug dose as that of the Tobi® deposited in the lungs, showed that the AUC values were found to be 1.6 times higher for Tobi® than for DPI formulations. So this evaluation confirmed the superiority of dry powder formulations in terms of drug deposition and reduced systemic exposure in comparison with the conventional comparator product, Tobi®.
Thus, these new and orginal tobramycin DPI formulations based on the use of very low excipient levels and presenting very high lung deposition properties, were shown to offer very good prospects for improving the delivery of drugs to the pulmonary tract and to the widest possible patient population.
II. INTRODUCTION
II. INTRODUCTION
II. 1. DRUG DELIVERY ROUTES TO THE LUNGS
A drug delivery system is defined as a formulation or a device or a combination of a formulation and a device that enables the introduction of a therapeutic substance into the body, and improves its efficacy and safety by controlling the rate, time and place of release of drug in the body. This process includes the administration of the therapeutic product, the release of the active ingredients by the product, and the subsequent transport of the active ingredients across the biological membranes to the site of action (Jain, 2008).
Drugs may be introduced into the human body by various anatomical routes. The choice of the route of administration depends on the disease, the effect desired, and the product available. As the systemic circulation supplies the whole lung, drugs to the pulmonary system may be given systemically by oral or parenteral delivery or administered directly to the organ affected by the disease.
II.1.1. Oral drug delivery
Historically, the oral route of drug administration has been the one most used for both conventional as well as novel drug delivery. The reasons for this preference are obvious because of the ease of administration and its widespread acceptance by patients. Major limitations of the oral route of drug administration are as follows (Jain, 2008):
1. Drugs taken orally for systemic effects have variable absorption rates and variable plasma concentrations, which may be unpredictable.
2. The high acid content and ubiquitous digestive enzymes of the digestive tract can degrade some drugs well before they reach the site of absorption into the bloodstream. This is a particular problem for aminoglycosides, ingested peptides and proteins.
3. The drug may be inactivated in the liver on its way to the systemic circulation.
4. As there is a dilution of the drug into the systemic circulation, high doses of drugs have to be administered in order to reach the therapeutic concentration in the lung, therefore causing enhanced systemic side effects. So the oral route may not be suitable for drugs targeted to specific organs.
II.1.2. Parenteral drug delivery
Parenteral literally means introduction of substances into the body by routes other than the gastrointestinal tract but in practice the term is applied to injection of substances by subcutaneous, intramuscular, intravenous, and intra-arterial routes. Parenteral administration of drugs is now an established part of medical practice and is the most commonly used invasive method of drug delivery. Major drawbacks of parenteral administration are as follows (Jain, 2008):
1. Injection is not an ideal method of drug delivery because it involves pain and patient compliance becomes a major problem.
2. As with the oral route, there is a dilution of the drug into the systemic circulation and it may not be suitable for drugs targeted to specific organs.
II.1.3. Pulmonary drug delivery
Drugs may be delivered directly to the lungs for local treatment of pulmonary conditions. Although simple inhalation devices and aerosols containing various drugs have been used since the early 19th century for the treatment of respiratory disorders, the interest in the use of the pulmonary route for systemic drug delivery is recent. Interest in this approach has been further stimulated by the demonstration of the potential utility of the lung as a portal for the entry of peptides and of the feasibility of gene therapy for cystic fibrosis. The advantages of pulmonary drug delivery are as follows (Jain, 2008):
1. Useful for local treatment and systemic distribution 2. A large surface area available for absorption
3. Close proximity to blood flow, highly vascularised tissue 4. Rapid absorption
5. Avoidance of first pass effect
6. Avoidance of the effects of gastric stasis and pH
7. Smaller doses required than by the oral route to achieve equivalent therapeutic effects.
II.2. CHARACTERISTICS OF THE LUNG
II.2.1. Anatomy of the airways
The airways may be viewed as a series of dividing passageways originating at the trachea and terminating at the alveolar sacs. The respiratory system consists of two tracts, upper and lower. The upper respiratory tract, consisting of the naso- and oropharynx and larynx, extends from the nostrils to the junction of the larynx and trachea. The oropharynx communicates with the mouth and serves as a passageway for food and air. The larynx connects the pharynx to the trachea, and conducts air to and from the lungs. The lower respiratory tract consists of tracheobronchial, gas-conducting airways and gas exchanging acini (Fig. 1).
The bronchial tree trunk begins with the trachea of the airways, which bifurcates to form main bronchi. As the trachea passes behind the arch of the aorta, it divides into two smaller branches: the left and right primary bronchi. Each primary bronchus divides into still smaller secondary bronchi, or lobar bronchi - one for each lobe of the lung. The secondary bronchi branch into many tertiary (or segmental) bronchi that further branch several times, ultimately giving rise to tiny bronchioles that subdivide many times, finally forming terminal bronchioles and respiratory bronchioles (Fig. 2). Each respiratory bronchiole subdivides into several alveolar ducts that end in clusters of small thin-walled air sacs called alveoli, which open into a chamber called the alveolar sac (Parks, 1994; Ross et al., 1995).
Figure 2: Representation of the bronchial tree (Learning the respiratory system, 2008)
In the classic model of the airways as described by Weibel (Weibel, 1963), each airway divides to form two smaller “daughter” airways. As a result, the number of airways at each generation is double that of the previous generation. The model proposes the existence of 24 airway generations in total, with the trachea being generation 0 and the alveolar sacs being generation 23. In reality, the branching is not perfectly symmetrical (Hickey and Thompson, 2004).
The walls of the primary bronchi, like the trachea, are supported by incomplete cartilage rings. In the lungs, the rings are replaced by small plates of cartilage of irregular shape that completely encircle the bronchus, giving the bronchi a cylindrical shape, in contrast to the ovoid shape with a flattened posterior wall of the trachea. With further branching, the cartilage plates gradually become smaller and fewer in number and the smooth muscles that surround the air passageways become more prevalent. Cartilage ultimately disappears at the point where the airway reaches a diameter of about 1 mm, whereupon it is designated a bronchiole (Parks, 1994; Ross et al., 1995).
The various levels of the airways may also be categorized functionally as being either conducting or respiratory airways. Those airways not participating in gas exchange constitute the conducting zone of the airways and extend from the trachea to the terminal bronchioles. The respiratory zone includes airways involved with gas exchange and comprises respiratory bronchioles, alveolar ducts, and alveolar sacs (Fig. 3) (Adjei et al., 1996;
Altiere and Thompson, 1996).
Figure 3: Representation of the alveoli (Learning the respiratory system, 2008)
In passing from the trachea to the alveolar sacs, two physical changes occur in the
decreases with the increasing generations - for example, a tracheal diameter might be of 1.8 cm compared to an alveolar diameter of 0.04 cm. This permits adequate penetration of air to the lower airways for a given expansion of the lungs. Secondly, the surface area of the airways increases with each generation, to the extent that the total lung area at the level of the human alveolus is in the order of 140 m² (Hickey and Thompson, 2004). Alveoli are the terminal air spaces of the respiratory system and are the actual site of gas exchange between air and the blood. About 100 million alveoli are found in each lung (Stone et al., 1992). Each alveolus is a thin-walled polyhedral chamber of approximately 0.2 mm in diameter (Fig.
4.A). Each alveolus is confluent with a respiratory bronchiole at some point, by means of an alveolar duct and an alveolar sac. Alveoli are surrounded and separated from one another by a thin connective tissue layer that contains numerous blood capillaries (Fig.4. B). The tissue between adjacent alveolar air spaces is called the alveolar septum. In traversing the air-blood barrier, gases in the alveolus must cross the alveolar epithelium, the capillary endothelium, and their basement membranes before reaching the blood, a distance in all of approximately 500 nm (Hickey and Thompson, 2004).
A B
Figure 4: A. SEM micrograph (2000x) of three alveoli in a secondary alveolar duct from the upper portion of the left main alveolar duct. The dark round openings are pores between alveoli. A macrophage in its spherical form is in the base of the alveolus in the upper right.
B. Close-up version of the alveolar wall. The red blood cells in a capillary are separated from the air by a very thin tissue barrier (Lung structure tour, 2008).
So, the alveolus is the principal site of gas exchange in the airways, a function compatible with the increased surface area, which promotes extensive and efficient diffusional gas exchange between the alveolar space and the blood in alveolar capillaries (Gehr et al., 1978).
II.2.2. Physiology of the airways
The epithelium of the airways is a continuous sheet of cells lining the luminal surface of the airways. The airway epithelium comprises a variety of cell types, the distribution of which confers different functions according to the airway region. Connecting adjacent epithelial cells are specialized tight junctional processes that limit the penetration of inhaled substances by the intercellular route (Hickey and Thompson, 2004).
Extending from the trachea to the terminal bronchus, the luminal surface of the airways is lined by ciliated cells, the most numerous of the cell types.
Mucus cells, which are similar in appearance to the intestinal goblet cells, are interspersed among the ciliated cells and also extend through the full thickness of the epithelium. Mucus, a viscous fluid containing mucin glycoproteins and proteoglycans, floats on a watery layer of pericilliary fluid and covers the luminal surface of the epithelium.
The secretions fulfil four important functions. Firstly, they protect the epithelium from becoming dehydrated. Secondly, the water in the mucus promotes saturation of inhaled air.
Thirdly, the mucus contains antibacterial proteins and peptides, such as defensins and lysozyme, that serve to repress microbial colonization of the airways. Fourthly, the mucus is involved in airway protection from inhaled xenobiotics or chemicals (Finkbeiner, 1999;
Schutte and Cray, 2002). The cilia, which appear as short hair-like projections from the apical surface, provide a coordinated sweeping motion of the mucus coat, providing a “mucociliary escalator” that serves as an important protective mechanism for removing small inhaled particles from the lungs (Philipps, 1981). Coordinated beating of the epithelial cilia propels the blanket of mucus toward the upper airways and pharynx, where the mucus may be
region such that movement in the smaller airways is slower than in the larger airways, a situation that arises from the proportionately larger number of ciliated cells and higher ciliary beat frequency in the larger airways (Gail and Lenfant, 1983). Mucus clearance from the airways is also enhanced by coughing, which rapidly propels the mucus toward the pharynx.
Failure to clear mucus from the airways as a result of ciliary disfunction or mucus hypersecretion (as may occur in cystic fibrosis or chronic bronchitis) can result in airway obstruction and infection. Such a situation may adversely affect the therapeutic activity of an inhaled drug by increasing the thickness of the mucus layer through which the drug must diffuse to reach its site of action (Hickey and Thompson, 2004).
The terminal bronchioles are lined with a simple cuboidal epithelium in which Clara cells are found among the ciliated cells. Clara cells are nonciliated cells that have a rounded or dome-shaped apical surface. They secrete a surface-active agent, a lipoprotein, to prevent luminal adhesion, particularly during expiration. A small amount of connective tissue underlies the epithelium, and a circumferential layer of smooth muscle underlies it in the conducting portions (Parkes, 1994; Ross et al., 1995).
Alveolar epithelium is composed of Type I and Type II alveolar cells and occasional brush cells. Type I pneumocytes are extremely thin squamous cells that line most of the surface of the alveoli - about 95% of the surface area. These cells are joined to one another and to the other cells of the alveolar epithelium - the Type II pneumocytes or septal cells, and the occasional brush cells, by zonulae occludentes. These tight junctions enable the cells to form an effective barrier between the air space and the components of the septal wall. In places, the cell membrane is invaginated to form pinocytic vesicles capable of ingesting macromolecules and particles that may be present in alveolar spaces, affording their transfer to the interstitium. Type II cells are cuboidal secretory cells interspersed among the Type I cells but tending to concentrate at septal junctions. They are the stem cells from which Type I cells differentiate and are replaced after injury. Type II cells are as numerous as Type I cells, but cover only 5% of the alveolar surface (Notter, 2000a; Notter 2000b). Their apical
cytoplasm is filled with lamellar bodies that are rich in phospholipids which are present in the surface-active agent, the surfactant (Parkes, 1994).
In fact, the surfactant is composed by weight of approximately 90% lipids and 10%
proteins. The lipids are characterized by an unusually high level of saturated fatty acid chains, such as the predominant dipalmitoylphosphatidylcholine (DPPC), which represents 40% in weight and contributes substantially to the unique properties of pulmonary surfactant (Fehrenbach, 2001). In addition to DPPC, lung surfactant contains unsaturated phosphatidylcholines (~35%), phosphatidylglycerol (~10%), phosphatidylinositol (~2%), phosphatidylethanolamine (~3%) and sphingomyelin (~2.5%). There is also a small amount (~3%) of neutral lipid, mainly cholesterol (Possmayer et al., 2001). The protein fraction comprises a highly variable amount of serum proteins and four apoproteins (SP-A, SP-B, SP- C and SP-D), which are associated with surfactant and contribute to its specific functions (Fehrenbach, 2001).
Lung surfactant acts to stabilize the lung alveoli during the respiratory cycle: it decreases the tendency for alveolar collapse during expiration by reducing surface tension in the terminal airways, and makes lung inflation during inspiration easier. Host defense is another function of alveolar surfactant that relies on the nature of SP-A and SP-D (Fehrenbach, 2001).
II.2.3. Blood supply
The lung has both a pulmonary circulation and a systemic circulation.The systemic circulation via bronchial arteries that branch from the aorta, supplies oxygenated blood and nutrients to the whole lung tissue other than the alveoli. It participates in air conditioning by assisting in the humidification and warming of inspired air in the trachea and bronchi. It also plays a central role in inflammatory conditions of the lung by contributing to mucosal oedema and delivery of inflammatory cells and mediators to the airways.
In contrast, the pulmonary circulation has a principal function of gas exchange of
dense, sheet-like capillary bed of the alveolar septum and is derived from the pulmonary artery that leaves the right ventricule of the heart. The blood is oxygenated and collected by pulmonary venous capillaries that ultimately coalesce to form the four pulmonary veins that return blood to the left atrium of the heart. Thus, 100% of the cardiac output flows through the pulmonary circulation, at a flow rate of 5l/min. It operates as a low-pressure, low- resistance vascular bed.
Both circulations anastomose at the level of the junction between the conducting and respiratory passages. Thus, drug delivered to the lower airways can enter the systemic circulation through absorption into the systemic circulation or into the alveolar capillaries of the pulmonary vascular bed (Ross et al., 1995; Altiere and Thompson, 1996).
II.2.4. Lung volumes
In healthy young males, the lungs have a total capacity of approximately 5900 ml.
About 1200 ml, called the residual volume, always remains in the lungs no matter how forced the expiration. During normal quiet respiration, about 500 ml of air moves into and out of the lungs, which is the tidal volume. The inspiratory reserve is the extra volume of air, approximately 3000 ml, that can be inspired in addition to the normal tidal volume. At the opposite extreme, additional air can be forced out (the expiratory reserve) and this has a volume of about 1200 ml. The vital capacity is the maximal amount of air that can be moved into and out of the lungs, and thus represents the sum of the inspiratory reserve, the tidal volume, and the expiratory reserve. In healthy young men the vital capacity is about 4700 ml.
All volumes are somewhat smaller in women because they tend to have smaller thoracic cages and smaller lung capacities (Parkes 1994, Ross et al., 1995).
II.3. PULMONARY DEPOSITION
II.3.1. Deposition mechanisms of inhaled particles
There are five mechanisms by which particles deposit in the respiratory tract. These are impaction, sedimentation, Brownian diffusion, interception and electrostatic precipitation.
Impaction is the inertial deposition of a particle onto an airway surface. It occurs principally at or near airway bifurcations, most commonly in extrathoracic and large conducting airways, where flow velocities are high and where rapid changes in the direction of bulk airflow often take place, generating considerable inertial forces. The probability of impaction increases with increasing air velocity, rate of breathing, particle size (> 5µm) and density (Martonen and Yang, 1996).
Gravitational sedimentation is an important mechanism for deposition of particles over 0.5 µm and below 5 µm in size in the small conducting airways where the air velocity is low. Deposition due to gravity is increased by large particle size and by longer residence times, and decreases with increasing breathing rate (Martonen and Yang, 1996).
Submicrometer-sized particles (especially those < 0.5 µm) acquire a random motion caused by the impact of surrounding air molecules. This Brownian motion may then result in particle deposition by diffusion, especially in small airways and alveoli, where bulk airflow is very low.
Interception is usually significant only for fibers and aggregates. For such particles, deposition can occur when a particle contacts an airway wall, even though its centre of mass might remain on a fluid streamline (Martonen and Yang, 1996).
Some freshly generated particles can be electrically charged during the mechanical generation of aerosols and may exhibit enhanced deposition, due to charges induced on the airway surface by these particles. However, this mechanism is a minor contributor to particle deposition (Lippmann and Schlesinger, 1984; Schlesinger, 1995)
In fact, the deposition patterns of inhaled particles may be expressed as functions of three classes of variables: ventilatory parameters, respiratory tract morphologies and aerosol characteristics (e.g., particle size, shape, and density). The efficiencies of the different deposition mechanisms of inertial impaction, sedimentation and diffusion can, in turn, be formulated in terms of these variables.
II.3.2. Influence of ventilatory parameters
Studies have demonstrated that total lung deposition can be markedly influenced by breathing profiles (Fig. 5) (Martonen and Katz, 1993). For instance, in the upper tracheobronchial tree the deposition of large (> 5 µm) particles can be primarily attributed to inertial impaction, whereas in the more peripheral airways it may be ascribed to sedimentation. By recognizing the effect of ventilatory parameters, deposition due to inertial impaction can be markedly enhanced in the upper lung by increasing the inspiratory flow rate, or, conversely, deposition in the lower lung can be promoted by increasing the duration of a postinspiratory pause (i.e. breath-holding time) (Martonen and Yang, 1996).
A B C
Figure 5: Calculated particle pulmonary deposition in function of A. the tidal volume, B. the breathing cycle and C. breath-hold time. Particles sizes are in micrometers (Martonen and Katz, 1993)
Quiet breathing is appropriate for an inhalation therapy aimed at targeting particles into the alveolar region. An increase in inhalation velocity may result in the development of turbulence, which tends to enhance deposition by impaction in the upper respiratory tract (Schlesinger, 1995). A slow inhalation rate (25 l/min) with breath holding showed maximal deposition of terbutaline sulphate compared to a faster rate (80 l/min) of inhalation (Newman et al., 1989).
Because the velocity distribution of air within the lung is determined by the tidal volume and breathing frequency parameters, the mass delivered to the alveoli can also be enhanced by increasing tidal volume, i.e., the volume of air inhaled during a single breath-hold of 2-6 s at the end of inspiration (Martonen and Yang, 1996). An inspiratory volume of 3000 ml with an inhalation rate of 60 l/min resulted in the highest deposition in the pulmonary region of the lung (Musante et al., 2002).
So, for a given patient (i.e. fixed morphology) and drug (i.e. prescribed aerosol), breathing is the only parameter that can be regulated
II.3.3. Influence of respiratory tract morphology
Among humans, individual variations in airway anatomy affect particle deposition in several ways: the diameter of the airway influences the displacement required by the particle before it contacts the airway surface; the cross section of the airway determines the low velocity for a given flow rate; and the variations in diameter and branching patterns along the bronchial tree affect the mixing characteristics between the tidal and reserve air in the lungs. There are also significant individual differences in respiratory tract anatomy, such as variations in the average alveolar-zone airspace size in humans (Lippmann and Schlesinger, 1984; Martonen and Yang, 1996).
Moreover, some diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and lung cancer may cause changes in the pulmonary tract by obstruction due to excessive production of mucus or constriction of the airways, influencing
II.3.4. Influence of aerosol characteristics: diameter
Particle size is one of the most important design variables in an aerosol formulation, along with shape, density, electrical charge and hygroscopicity. Aerodynamic diameter is the most appropriate measure of aerosol particle size because it relates to particle dynamic behaviour and describes the main mechanisms of aerosol deposition as both gravitational settling and inertial impaction, depending on aerodynamic diameter. In order to reach the lower respiratory tract and optimize pulmonary drug deposition, dry powder aerosols need to present aerodynamic diameters of between 1 and 5 µm (Zanen et al., 1994; Zanen et al., 1995; Zanen et al.1996; Elversson et al., 2003). Particles larger than 5 µm usually deposit in the oropharynx, from which they are easily cleared. In contrast, particles smaller than 0.5 µm may not deposit at all, since they move by Brownian motion and settle very slowly (Heyder et al., 1986; Bosquillon et al., 2004).
The aerodynamic diameter (Daer) is defined by the equation:
Daer = Dgeo √(ρp/ρ0χ) (1)
where Dgeo is the geometric diameter, ρp and ρ0 are particle and unit densities, and χ is the dynamic shape factor.
Pharmaceutical powders are rarely spherical, and shape factors are dimensionless measures of the deviation from sphericity. The dynamic shape factor is the ratio of the actual resistance force experienced by a nonspherical falling particle to the resistance force experienced by a sphere having the same volume (Hinds, 1999). So the aerodynamic diameter can be decreased by decreasing the particle size, decreasing particle density, or increasing the dynamic shape factor.
Aerosol particle design therefore involves two basic strategies. Either particles are made with standard unit density with a geometric size in the 1-5 µm range, or they are created with a non-standard density, and therefore with geometric sizes outside the standard range (Edwards and Dunbar, 2002) (see part II.5.1.5, p 56). As an example, particles exhibiting a high respirable fraction with mean geometric diameters ranged between 3 and 15 µm and tap densities between 0.04 and 0.6 g/cm³ could be produced (Vanbever et al., 1999).
II.4. DELIVERY DEVICES
A good delivery device has to generate an aerosol of suitable size, ideally in the range 0.5-5 µm, and a reproducible drug dosing. It must also protect the physical and chemical stability of the drug formulation. Moreover, the ideal inhalation system must be a simple, convenient, inexpensive and portable device (Dolovich et al., 2005).
Inhaled drug delivery devices can be divided into three principal categories: nebulizers, pressurized metered-dose inhalers (MDIs) and dry powder inhalers (DPIs), each class with its unique strengths and weaknesses.
II.4.1. Nebulizers
Nebulizers have been used in inhalation therapy since the early 19th century.
Marketed respiratory solutions are generally composed of drug dissolved in aqueous, isotonic solvent systems that may contain preservatives to reduce microbial growth. There are two traditional devices: air-jet and ultrasonic nebulizers.
range for inhalation. Only smaller droplets with less inertia can follow the streamlines of the air and pass the baffle (Le Brun et al., 2000). Approximately 50-60% of the particles produced For a typical jet nebulizer (Fig.6), compressed
air passes through a narrow hole and entrains the drug solution from one or more capillaries mainly by momentum transfer. The complex liquid break-up process largely depends on the nozzle design and is usually a combination of turbulent rupture of the instable liquid column and secondary droplet break-up. Large droplets impact on one or more baffles in order to refine the droplet size distribution to the required
Figure 6: Schematic presentation of a jet nebulizer (Le Brun et al., 2000)
Alternatively, ultrasonic nebulizers use a high frequency vibrating plate to provide the energy needed to aerosolize the liquid. The frequency of the vibrating piezoelectric crystal determines the droplet size for a given solution. Approximately 70% of the particles produced present sizes of between 1 and 5 µm. Nevertheless, heat resulting from frictional forces induced by movement of the transducing crystal may be detrimental to thermolabile formulations.
In fact, early nebulizers were cumbersome and unreliable. Currently, they have the ability to generate small droplets capable of penetrating deeply into the lung, they have high dose delivery capacity (e.g. antibiotics), and some are miniaturized, high-output devices.
New developments in liquid spray delivery devices include the use of piezo-electric atomization, high pressure micro-spray nozzle systems and electrostatic generation of aerosol clouds (Smith, 2002). Moreover, coordination between aerosol generation and breathing, which is required for successful metered-dose inhaler use (see below part II.4.2, p 33), is not essential for nebulizers, making them useful for elderly and very young patients (Clay and Clarke, 1987).
Some of the most commonly available nebulizers on the market are: Ventolin® (Salbutamol, ß2-mimetic bronchodilator), Bricanyl® (Terbutaline, ß2-mimetic bronchodilator), Atrovent® (Ipratropium, anticholinergic bronchodilator), Pulmozyme® (Dornase alpha, mucolytic) and Tobi® (Tobramycin, antibiotic).
But nebulization has many well-documented disadvantages, including extended administration time, high cost, low efficiency, poor reproducibility and great variability, risk of bacterial contamination and constant cleaning requirements, and sometimes the need for bulky compressors or gas cylinders (Newhouse et al., 2003). Moreover, in some cases, the presence of preservatives such as sodium metabisulfite, benzalkonium chloride and ethylene diamine tetraacetic acid (EDTA) has caused coughing and bronchoconstruction (Dalby et al., 1996).
One of the principal problems of nebulizers is that the device includes a large “dead volume”
of solution. A large fraction of the amount (up to 50%) can thus remain trapped in the
apparatus. Moreover, the aerosolized drug is generated continuously, leading to drug waste (Dalby et al., 1996). With a continuously working compressor (continuous droplet generation), part of the aerosol cloud may be wasted into the environment through the vent when the patient stops or interrupts inhalation or does not inhale fast enough. The amount inspired is equivalent, more or less, to half of the delivered amount. Of this inhaled amount, it is still necessary to remove a fraction of particles that are not in the “respirable range”. In conclusion, the pulmonary fractions obtained using a nebulizer may vary from 2-10% of the nominal dose. As an example, 2.5 ml of Pulmozyme at 1 mg/ml is delivered by a jet nebulizer with an estimated delivery efficiency of 10% (Cipolla et al., 1994).
Reduction of the waste by at least 50% of the nebulized dose may be achieved by so- called breath-assisted open vent nebulizers. The vent has a flexible membrane (valve) that opens only during inhalation. Meanwhile, a similar membrane in the outlet tube closes the route for exhalation. When the patient does not inhale, both valves are closed in order to prevent waste of the produced drug aerosol to the environment (Le Brun et al., 2000).
Nevertheless, it is a known fact that patient compliance is poor in inhalation therapy.
There are only a few evaluation studies that refer to compliance. In a study on children with respiratory diseases a compliance of 47.6% with the prescribed inhalation therapy was found (Schöni, 1993), whereas another study found a mean compliance of only 56.8% for adult patients on inhalation therapy (Cochrane, 1997). This low compliance is understandable because it usually takes a lot of time and energy to inhale the prescribed medication on a daily basis. Another aspect of the daily routine is the maintenance of the nebulizer. Cleaning and disinfection is necessary in order to prevent contamination of the nebulizer and subsequent possible infections (Reychler et al., 2007).
